Production and Characterization of Phytase from Streptomyces Luteogriseus R10 Isolated from Decaying Wood Samples
Total Page：16
File Type：pdf, Size：1020Kb
INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 14–453/2015/17–3–515–522 DOI: 10.17957/IJAB/17.3.14.453 http://www.fspublishers.org Full Length Article Production and Characterization of Phytase from Streptomyces luteogriseus R10 Isolated from Decaying Wood Samples Magda Mohamed Aly1, 2, Sanaa Tork1, 3, Saleh Mohamed Al-Garni1* and Saleh Abdulla Kabli1 1Biology Department, Faculty of Science, King Abdulaziz University, Saudi Arabia 2Botany Department, Faculty of Science, Kafr El-Sheikh University, Egypt 3Microbial Genetics Department, National Research Center, Dokki, Cairo, Egypt *For correspondence: [email protected] Abstract Microorganisms are the richest source of phytase which catalyze hydrolysis of phytate to myo-inositol and phosphate. 50 Actinomycete isolates were isolated from heated soil, sand, wastewater, animal faces and some plant ecological wastes on Starch nitrate agar containing 15 µg / mL of antibiotics (Tetracycline + Amphotericin B, w/w). Out of 50 Actinomycetes isolates, 20 isolates (40%) produced extracellular phytase enzyme on solid medium containing wheat bran as carbon source. In liquid medium, the phytase activity was measured as U/mL and the most active isolate in phytase production was R10. Using morphological, physiological and biochemical studies, it was identified as Streptomyces sp. Using 16S rDNA analysis, it was identified as S. luteogriseus R10. Growth in medium containing 1% Na-phytate (pH 6.5) at 40°C for 7days increased phytase production. The maximum phytase activity was achieved using wheat bran, straw, rice husk and hay after seven days of incubation at 40°C but low activity was obtained using Sawyer and baggage as a carbon source. The molecular weight of the purified phytase is 65 kDa and it exhibits optimum activity at pH 5 and 45°C. All tested metal ions at 10 mM enhanced phytase activity except Ba2+, Co2+, Cu2+, Ag, Fe3+ and Hg2+. Improvement of phytase production was carried out using protoplast fusion between S. luteogriseus R10 and S. niveus MM1. Fusant F7 was the best phytase producer (3 time higher) compared to its parents. © 2015 Friends Science Publishers Keywords: Phytase; Streptomyces; Protoplast; Fusant; Phytic acid Introduction low level of phytase in primitive natural microbes. For humans and animals, legumes, cereals and oilseed crops Materials and Methods are the main source of nutrients, which contain phytic acid as storage form of phosphorus (Reddy et al., 1989). To Bacterial Isolation satisfy phosphorus requirement, inorganic phosphate is added to monogastric animals diets because they cannot Soil, sand, plant materials, wastewater and plant waste metabolize phytic acid present in their diet. In monogastric products from farms in Huda Al Sham, Saudi Arabia were animals, phytic acid is chelating various metal ions collecting and transported in sterile plastic bags to the including calcium, copper and zinc (Graf, 1983). Therefore, Microbiology lab. Serial dilutions were prepared and about phytic acid hydrolysis into less-phosphorylated myo-inositol 0.1 mL of each sample was spread on starch nitrate medium derivatives using phytase is of great interest. Phytase can be (Shirling and Gottlieb, 1966) which composed of g/L: 20 used to improve the nutritional value of feed and/or to Starch, 1.0 KH2PO4, 0.5 MgSO4.7H2O, 0.01 FeSO4.7H2O, decrease the amount of phosphorus excreted by animals. 3.0 CaCO3, 2.0 KNO3, 0.5 NaCl and 1 mL trace salt Phytase enzyme produced by bacteria is extracellular solution) and for solid medium preparation, 20 g/L agar was which are more appropriate than the intracellular added. Filter sterilized mixture of phytase produced by yeast in breaking down phytic acid Tetracycline+Amphotericin B (w/w) was added at (Konietzny and Greiner, 2004). The aim of the present concentration 15 µg/mL to the prepared medium (Aly et al., study was isolation and identification of thermo-stable 2011b). Incubation of the plates was at 37°C for 7 days and phytase producing bacterium and factors affecting the purified colonies were streaking on ISP2 medium production were studied. Phytase was purified and composed of g/L: 4 yeast extract, 4 glucose, 10 malt characterized. Enhancement of enzyme production was extract, 2 CaCO3 and 20 agar and maintained at 4°C carried out using protoplast fusion to solve the difficulty of until used. To cite this paper: Aly, M.M., S. Tork, S.M. Al-Garni and S.A. Kabli, 2015. Production and characterization of phytase from Streptomyces luteogriseus R10 isolated from decaying wood samples. Int. J. Agric. Biol., 17: 515‒522 Aly et al. / Int. J. Agric. Biol., Vol. 17, No. 3, 2015 Bacterial Growth and Screening Conditions precipitate at 10,000 rpm was dissolved in phosphate buffers (pH 7), dialyzed against the same buffer for 2 days Several Actinomycete isolates were screened on modified (El-Sabbagh et al., 2003), concentrated under vacuum, LB with wheat bran medium (LBWB medium). LB medium applied to a column (30 × 1.5 cm) of diethyl aminoethyl composed of (g / L) 10 g tryptone, 10 g NaCl and 5 g yeast cellulose (DEAE cellulose) and eluted using 1 M NaCl in extract in addition to 50 g of wheat bran, pH was adjusted to phosphate buffer (80 mL / h). The eluent was collected in 5 pH 7.0. Agar (20 g / L) was added, when solid agar medium mL fractions. The active fractions with phytase activity was needed. In liquid broth medium, the bacterial isolates were collected and concentrated under vacuum. The with phytase activities were cultivated in 50 mL LBWB concentrate was applied to carboxymethyl-cellulose broth medium in 250 mL Erlenmeyer flasks. Each flask was followed by Sephadex G75 column and elution was carried inoculated with 2mL (4×106 CFU∕mL) of bacterial out using phosphate buffer. The active fraction was suspension, previously grown at 37°C for 2 days at 200 rpm collected, lyophilized and was analyzed. Gel electrophoresis in starch nitrate broth medium. The inoculated flasks were was carried with 15% SDS polyacrylamide electrophoresis maintained for 5 days at 37°C on a rotary shaker (200 rpm). at room temperature where 20‒30 μL (40 μg / well) from After centrifugation at 10,000 rpm for 15 min, cell-free the protein standard (Merck) were applied and gel was supernatant was for phytase assay. stained with Coomassie brilliant blue R-250. Phytase Assay Characters of the Pure Phytase Enzyme Cell-free supernatant was used for determination of phytase Using the standard phytase assay conditions (Chi et al., activity by measuring the amount of phosphorous released 2008), effect of different pH values, 3‒9, on phytase was from Na-phytate, which develops a color with ammonium determined after the pure enzyme suspension in 0.2 M molybdate. The color density was quantified acetate buffer (pH 3‒6) or 0.2 M Na2B4O7.10 H2O / H3BO3 spectrophotometrically at 700 nm (Chi et al., 1999). Under buffer (pH 7‒10). Effect of temperature ranged from 20- assay conditions, one phytase unit was the amount of the 70ºC on the purified enzymes was determined in the enzyme, released of 1 nM of inorganic phosphate / min. selected buffer. The pre-incubated enzyme at 37°C was used as a reference to calculate activity. Effect of some additives Optimization of Phytase Production Process incorporated in the reaction mixture including some metal ions and EDTA on the enzyme assay was studied and the In liquid medium, the effect of different factors on growth relative activities were compared with the activity obtained (optical densities at 540 nm) and phytase production was for control (without additive) (El-Sabbagh et al., 2003). determined (El-Sabbagh et al., 2003). LB broth medium with different concentrations of Na-phytate was prepared. Characterization of the Selected Actinomycete Isolate The inoculated flasks were incubated at 37ºC and 200 rpm (Taxonomical Studies) for 5 days. Effect of different incubation temperatures (20- 50°C) and medium pH (6.0-8.0) were determined after 5 The selected phytase producing actinomycete isolate was day of growth in LB medium with 1% Na-phytate. At the characterized and identified. It was grown on starch nitrate end of growth period, growth and phytase were measured. agar medium for fresh prepared culture. Gram and Effect of different incubation periods ranging from 1 to 7 endospore stains were carried out and bacterial morphology days of growth in modified LB medium (pH 6.5) at 40°C was conducted using light and electron microscopy (XL30- and 200 rpm was determined. All the experiments were ESEM environment scanning electron microscopy). carried out in triplicate and averages were reproduced. Physiological and biochemical characters in addition to chemical analysis of the whole cell sugar composition and Phytase Production Using Various Waste Products type of the diaminopimelic acid isomer were determined as described by Aly et al. (2011a, 2012) and Hasegawa et al. Some agriculture waste products were collected, dried, (1983), respectively. Cell fatty acids and phospholipids were powdered and sterilized using the autoclave. They were extracted and determined using gas chromatography and used as carbon source as described by Lanciotti et al. (2005) two-dimensional thin-layer chromatography (Butte, 1983; and phytate hydrolysis was determined after 7 days of Hoischen et al., 1997). incubation at 40°C and 200 rpm. Phylogenetic Analysis of 16S rDNA Sequence Enzyme Purification and Molecular Weight Determination QIAamp DNA Mini Kit was used for genomic DNA extraction of the selected isolate R10. The forward primer 5' Proteins in the culture filtrate were collected after 80% AGTTTGATCATGGTCAG-3' and reverse primer 5' Ammonium sulfate precipitation at 4°C. The collected GGTTACCTTGTTACGACT 3' were designed (Weisberg 516 Improved Phytase Production by Actinomycetes R10 / Int. J. Agric. Biol., Vol. 17, N0. 3, 2015 et al., 1991) and 16S rDNA gene was amplified, sequenced and the DNA sequence was compared to the GeneBank database.